Reproducibility and Replication
After years of experience with E. coli arrays, including
the GenoSys membranes, we can now routinely use them for robust
gene expression profiling that properly reflects “biological reality”.
Replication is the key! We routinely use 3 or more cultures for
typical laboratory (flask or testtube) experiments – biological
replicates. We routinely obtain two technical replicates for each
RNA sample (2 separate labeling/hybridizations, same RNA). We treat
the two spots on the array as independent measurements and normalize
arrays by expressing the intensity of each spot as a percentage
of the sum of all spot intensities. In flask and testtube cultures,
the correlation coefficients for all spots is usually >0.98 for
technical replicates and >0.95 for biological replicates. We
occasionally obtain results that must be thrown out, or averaged
with additional replicates. The most dramatic improvement in culture-to-culture
reproducibility came when we switched from flasks to a fermenter.
We now grow almost all of our batch cultures in a 1liter volume
on a Braun BiostatB with constant conditions and pH-stat. The biological
reproducibility of technical replicates from different cultures
by this method is always >0.99 (correl).
Top figure: Technical replicates of one RNA sample hybridized to
a pair of “matched” membranes.
Bottom figure: Biological replicates of two cultures: E. coli
MG1655 grown in fermenter on MOPS-glucose minimal medium at pH7.4,
aerobically, 37oC. Data represent average of 2 hybridizations.